Coherent Raman Scattering Microscopy - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 125

that often do not offer chemical contrast. Applications of CARS tissue imag-
ing with subcellular resolution include the study of in vivo mouse skin [26],
axonal myelin in both ex vivo and in vivo spinal nerve tissues [96, 87, 97, 101],
lipid storage in a living Caenorhabditis elegans nematode [98], brain tissue
structures [99], arterial tissues [100, 43], collagen sheaths around internal or-
gans and muscles [102], liver tissue [103], and the localization of metal oxide
nanoparticles in fish grill lamellae [115]. In the following, we first discuss an
exemplary demonstration of the full potential of CARS imaging in live tissue
and then describe an example of multimodal CARS microscopy in a spectral
focusing implementation that allows simultaneous imaging of three endoge-
nous nonlinear signals.
Cheng and coworkers [96, 97, 116, 117] have demonstrated real-time CARS
imaging of neuronal myelin under physiological conditions. Myelin sheaths
that surround axons in the central nervous system are formed of wrapped cell
membranes. The high density of CH 2 groups in myelin leads to a strong and
directional CARS signal at 2840 cm − 1 that allows overcoming the sensitivity
limitations of Raman microscopy and thus facilitates high-speed imaging of
live spinal tissues. Figure 6.6A shows a CARS image of normal myelin sheath
when focused into the equatorial plane of axons. E cient CARS generation
is observed when the linearly polarized pump and Stokes laser beams are in
the direction parallel to the vertical orientation of axons, whereas horizon-
tally polarized beams show weak CARS contrast. This photoselection effect
[69] reveals the preferential vertical alignment of the symmetry axis of the
CH 2 groups in the equatorial plane of the myelin (Fig. 6.6C). The ratio be-
tween the two polarization-resolved CARS image pixel intensities allows the
determination of the degree of ordering of the myelin lipids. The image in
Fig. 6.6B exemplifies the ability to monitor chemically induced degradation
and disorders of myelin in spinal tissues [116]. Extensive myelin swelling char-
acterized by a decrease in CARS contrast was observed after incubation of the
sample with lysophosphatidylcholine (Lyso-PtdCho). Furthermore, the com-
parison with the polarization-resolved images of normal myelin sheath reveals
a lack of a pronounced photoselection effect, indicating an increased disorder
of lipid orientations upon myelin swelling. As this experiment demonstrates,
CARS imaging facilitates the real-time characterization of the molecular dis-
tribution and the orientation of lipids in live spinal tissues under physiological
conditions. It therefore offers the opportunity to study pathological pathways
in neurological diseases.
The chemical specificity of CARS microscopy is readily combined with
other nonlinear optical image contrast mechanisms, such as two-photon flu-
orescence (TPF), SHG, and THG, resulting in a multimodal CARS mi-
croscopy [88, 118, 117, 43]. In multimodal nonlinear optical imaging, TPF,
SHG, and THG signals all benefit from the use of femtosecond laser pulses of
high peak intensities, whereas the contrast and chemical selectivity of CARS
benefits from the use of picosecond (narrow-bandwidth) pulses (see discus-
sion in Sect. 6.2.3). As demonstrated by Pegoraro et al. [43], this apparent